1042

recognised premalignant lesions elsewhere in the bodymay take years to become malignant; and few gyneecolo-gists can have carefully followed enough cases of leuco-plakia for long enough to estimate the hazard of malignantchange.

PRECIPITIN TESTS IN VIRUS RESEARCH

PRECIPITIN tests have not been used extensively in

virology, partly because it has been difficult to obtain

enough antigen to produce a visible precipitate, and partlybecause the possibilities of the technique have beenneglected. Precipitin techniques, especially those employ-ing diffusion through agar gel, are very useful in immuno-logical analysis. These techniques have been well reviewedby GrowledThe most important application of these techniques in

virology is in the diagnosis of smallpox. The test employ-ing agar-gel diffusion was devised by Dumbell andNizamuddin 2 and depends on detection of antigen incrust extracts or vesicle fluids by formation of a precipitinline with vaccinia antiserum in a double-diffusion test.The double-diffusion technique is much superior to theprecipitin test in tubes first described by Gordon 3 in1925, and to the complement-fixation test, since fusionof the line given by the test sample with the vacciniacontrol is convincing evidence of identity. This methoddoes not distinguish between smallpox and vaccinia sincethese viruses are so closely related antigenically. Chicken--pox-and zoster infection can also be established in thelaboratory by demonstration -of the specific antigen invesicle fluid, and again no difference between these twoviruses can be detected.4

Serological diagnosis in virus diseases is, unfortunately,rarely possible by identification of the specific virus antigen;more often it depends on demonstrating a rise in titre ofviral antibodies. The advantages of an agar-gel diffusiontechnique over more conventional serological techniquesare illustrated by studies on the diagnosis of diseasesassociated with enterovirus infection. Le Bouvier 5 showedthat D and C antigens of poliovirus can be distinguishedby gel-diffusion precipitin tests. When complement-fixation or precipitin tests in fluid media are employed,confusion can arise because more than one antigen-antibody system may contribute to the observed result.The diagnostic value of the detection of specific

precipitins in human sera has been shown by Schmidt andLennette 6 for Coxsackie B and A9 viruses. They foundthat the gel-diffusion precipitin method of diagnosis,which depends on the presence of antibodies rather thana titre rise, was much more sensitive than complement-fixation or neutralisation tests for these viruses, exceptCoxsackie B2. Thus of 87 cases yielding a Coxsackie virus,84 were diagnosed by the precipitin test, whereas onlyabout 50% of cases of Coxsackie infection show a diag-nostic titre rise by the complement-fixation or neutralisa-tion technique. In addition, of 86 patients who failed toyield a virus, 23 had precipitins; presumably thesediseases were associated with recent Coxsackie-virusinfections. The special value of the precipitin test in

agar was revealed in these experiments because a group-specific antigen was present in the majority of sera,

1. Crowle, A. J. Immunodiffusion. New York, 1961.2. Dumbell, K. R., Nizamuddin, Md. Lancet, 1959, i, 916.3. Gordon, M. H. Spec. Rep. med. Res. Coun., Lond. 1925, no. 98.4. Taylor Robinson, D., Rondle, C. J. M. Brit. J. exp. Path. 1959, 40, 517.5. Le Bouvier, G. L. ibid. p. 452.6. Schmidt, N. J., Lennette, E. H. J. Immunol. 1962, 89, 96.

and only the type-specific precipitin line had diagnosticsignificance. Tests in fluid media would not have

separated the two antigen-antibody systems. Coxsackie B2,for example, gave only one line which was almost certainlya fused group and specific precipitate. Selzer,’ too, hasshown that precipitin tests in agar can be used for thediagnosis of recent poliovirus infection-a possibility firstclearly shown by Le Bouvier.8 She observed that the merepresence of strong lines or double lines indicated recentinfection. The presence of double lines presumablydenotes the presence of both C and D antibodies, but thispoint was not specifically examined.

Wilson Smith et al. and Schmidt and Lennette 10found that precipitin tests in fluid media could be usedfor poliovirus diagnosis. Precipitins declined quickly inconvalescence so that the presence of antibodies had

diagnostic significance. Roizman et al. 11 found that Cantibodies appear and decline earlier than D antibodies

according to the complement-fixation test, but it is notclear which antigen was measured in the precipitin tests.It is clearly important to differentiate between these

antigens, and this can be done conveniently in agar-gelprecipitin tests with suitable control antigens.5 It has

always been easy to obtain pure C preparations by heat,but only recently has pure D antigen become easilyobtainable. 12

Precipitin lines in agar have also been demonstratedwith convalescent influenza serum,13 whereas no precipitinshave been detected in these sera by tests in fluid media."Lack of precipitation in fluid media may be due to thepresence of blocking antibodies that combine with virus:these have been demonstrated by Wilson Smith 15 in

poliovirus antigen-antibody systems.Agar-gel precipitin techniques are very versatile methods

of antigenic analysis, especially when supplemented byelectrophoresis. One interesting application has been inthe analysis of adenovirus antigens. This virus is too largeto diffuse through agar, but three other viral antigens havebeen revealed by immuno-electrophoresis.16 They arecalled A, B, and C. A is the group-specific antigen, and Cthe type-specific antigen, while B is the factor responsiblefor the early cytopathic effect produced by adenovirus.Precipitin tests in agar have also been used for the diagnosisof adenovirus infections.17 Nine antigen precipitin lineshave been described in cowpox preparations.l8 Anotherapplication has been in the assay of the amount of antigenin poliovirus preparations by Le Bouvier,5 and this methodhas been exploited by Beale and Mason 19 for poliovaccinepotency determinations. This technique had earlier beensuggested by Brown and Crick 20 as a means of assessingthe potency of foot-and-mouth-disease vaccine.

Clearly, precipitin tests in agar are a valuable tool whichis beginning to be widely exploited in virology. Any workerwith a problem which may be solved by the application ofthese techniques can be recommended to read Crowle’smonograph,’ which is a mine of technical information.7. Selzer, G. J. Hyg., Camb. 1962, 60, 69.8. Le Bouvier, G. L. J. exp. Med. 1957, 106, 661.9. Smith, W., Boissard, G. P. B., Churcher, G. M., Parker, L. Lancet, 1959,

ii, 586.10. Schmidt, N. J., Lennette, E. H. Amer. J. Hyg. 1959, 70, 51.11. Roizman, B., Mayer, M. M., Rapp, J. H. J. Immunol. 1958, 8, 419.12. Henle, W., Henle, G., Hummeler, K., Lief, F. S. J. Pediat. 1961, 59,

827.13. Jensen, K. E., Francis, T. J. Immunol. 1953, 70, 321.14. Belyavin, G. Brit. med. Bull. 1959, 15, 193.15. Smith, W. Virology, 1961, 13, 280.16. Pereira, H. G., Allison, A. C., Farthing, C. P. Nature, Lond. 1959, 183,

895.17. Pereira, M. S., Pereira, H. G., Allison, A. C. Lancet, 1959, i, 551.18. Rondle, C. J. M., Dumbell, K. R. J. Hyg., Camb. 1962, 60, 41.19. Beale, A. J., Mason, P. J. ibid. p. 113.20. Brown, F., Crick, J. Virology, 1958, 5, 133.